Obesity is a public health issue in the United States with more than one third of the adult population being identified as obese. Obesity in children is also on the rise. Obesity is also associated with an increased risk of a variety of co-morbid conditions such as diabetes, atherosclerosis and hypertension. Obesity also is one of the leading risk factors for metabolic syndrome. Metabolic syndrome is a group of five risk factors that increase an individual's risk for heart disease and other health problems such as diabetes and stroke. The five conditions or risk factors are high blood pressure, low HDL cholesterol levels in blood, large waistline, high triglyceride levels in blood, and high fasting blood sugar. Individuals with three or more of these conditions are diagnosed with metabolic syndrome. Metabolic syndrome is becoming an increasingly common diagnosis as the obesity rates rise in the United States, with health professionals predicting that, sometime in the near future, metabolic syndrome may overtake smoking as the leading risk factor for heart disease. It is known that one of the diseases that commonly develops in patients with metabolic syndrome is type II diabetes; in turn, the number one cause of death of patients with type II diabetes is atherosclerosis, a disease that causes plaques to build up in the arteries and eventually lead to heart attacks and stroke. As a result, there is a great deal of interest in identifying new targets for development of therapies to prevent and treat obesity, metabolic syndrome, and the conditions that can develop as a result, such as atherosclerosis.
Acyl-CoA:Cholesterol Acyltransferase (ACAT) converts free cholesterol to cholesterol ester, and is one of the key enzymes in cellular cholesterol metabolism. Two ACAT genes have been identified which encode two different enzymes, ACAT1 and ACAT2 (also known as SOAT1 and SOAT2). While both ACAT1 and ACAT2 are present in the liver and intestine, the cells containing either enzyme within these tissues are distinct, suggesting that ACAT1 and ACAT2 have separate functions. Both enzymes are potential drug targets for treating dyslipidemia and atherosclerosis.
WO 2003/088962 discloses combining administration of a PPARα/γ dual agonist compound with a compound that inhibits ACAT activity in order to treat lipid disorders as well as obesity. Nowhere does the application teach use of an ACAT inhibitor alone, or use of inhibitors specific for one ACAT isoform. Therefore, this application fails to teach that inhibiting ACAT1 specifically in macrophages would be beneficial in the treatment of obesity and/or atherosclerosis.
WO 2004/111084 discloses use of peptides that enhance cholesterol ester hydrolase activity (CEH) or inhibit ACAT activity to treat atherosclerosis. The application teaches use of peptides that target macrophage ACAT enzymes. WO 2006/0257466 also discloses combining CEH enhancers and ACAT inhibitors that are targeted to macrophages in the treatment of atherosclerosis. WO 2006/105666 teaches administering macrophage-targeted formulations that target modulation of ACAT activity alone for the treatment of atherosclerosis. The inhibitors mentioned were not isoform-specific, and no data were provided demonstrating that inhibition of ACAT1 specifically in macrophages would be beneficial for treatment of atherosclerosis or obesity.
U.S. Pat. No. 6,121,283 discloses treatments for obesity that involve administration of ApoB/MTP inhibitors as the primary agents, and then potentially combining these agents with inhibitors of a variety of targets including ACAT.
WO 2009/081957 discloses use of a drug, beauveriolide, to inhibit activity of ACAT2, but not ACAT1, as a treatment for lipid disorders as well as obesity. KR 1020030011474 discloses use of a drug, panaxynone A, a polyacetylene compound, to suppress activity of ACAT. Also taught is the use of this drug to suppress obesity by reducing body weight.
U.S. Patent Application No. 2011/0184173 and EP 2228376 disclose administration of pyripyropene derivatives for inhibiting activity of ACAT2, but not ACAT1. Also taught is the use of these compounds to treat a variety of conditions including disorders of lipid metabolism and obesity.
Stahlberg et al. (1997. Hepatology 25:1447-1450) studied hepatic cholesterol metabolism in obese and non-obese subjects. They reported increases in ACAT activity in obese subjects and attributed these increased levels of ACAT activity to higher concentrations of microsomal free cholesterol. Obese subjects also exhibited large increases in cholesteryl esters in liver. Nowhere does this paper teach or suggest that obesity could be treated or prevented by administration of ACAT inhibitors.
Turkish and Sturley (2007. Am J. Physiol. Gastrointest. Liver Physiol. 292:G953-G957) discuss the role of neutral lipid metabolism in the pathogenesis and/or treatment of diseases including obesity. As discussed in this review paper, the apparent redundancy in neutral lipid synthesis is advantageous, and marked changes in lipid homeostasis arise when expression of acyl CoA:diacylglycerol acyltransferase (DGAT), in particular, is altered. The authors speculate that isoform-specific inhibition of DGAT1, DGAT2, ACAT2 or the acyl-CoA wax alcohol acyltransfereases (AWATs) may be effective and non-toxic therapeutics for type II diabetes and obesity, for example. No specific data are provided showing any effect of ACAT1 or ACAT2 inhibition to prevent or treat obesity. This paper also failed to disclose any role for modulating macrophage-specific ACAT1 activity as a method of treating obesity and/or atherosclerosis.
Tomoda and Omura (2007. Pharmacol. & Therapeut. 115:375-389) discussed potential therapeutics for obesity and atherosclerosis. The class of compounds disclosed was neutral lipid metabolism inhibitors isolated from microorganisms. Both DGAT and ACAT inhibitors were discussed, including both synthetic compounds and compounds of microbial origin. The synthetic ACAT inhibitors included pactimibe, avasimibe, Wu-V-23 and CL-283,546. The microbial origin compounds included pyripyropene, pyripyropene derivatives A through D, purpactin A, purpactin B, purpactin C, glisoprenin A, glisoprenin B, terpendole C, terpendole D, beauvericin, spylidone, and sespendole. As discussed, the authors indicated that the selectivity of ACAT inhibitors toward ACAT1 and ACAT2 is only partially understood. The authors concluded that DGAT and ACAT have been identified as potential therapeutic targets. DGAT1 knockout mice are reported to be resistant to obesity, while DGAT2 knockout mice die soon after birth, making DGAT1 a more promising target for anti-obesity therapeutics. The only actual data discussed with respect to ACAT inhibition, however, related to the activity of ACAT inhibitors in the treatment of atherosclerosis, not obesity itself.
The role of ACAT proteins in promoting or protecting against atherosclerosis, co-morbidity associated with obesity, has been investigated. Rudel et al. (2005. Arterioscler. Thromb. Vasc. Biol. 25:1112-1118) proposed that decreased activity, i.e., inhibition, of ACAT2 is atheroprotective. These authors also predicted that the role of ACAT1 activity is to destabilize cellular membrane function and promote macrophage cell death. The prediction made by these authors (i.e., decreased ACAT1 activity in macrophages can lead to macrophage cell death in vivo) was based on in vitro data only. The authors suggested that ACAT2 is more important as a target for treating coronary artery disease, but provide no in vivo data on the role of ACAT1. In a review article, Lopez-Farre et al. (2008. Cardiovasc. Ther. 26:65-74) proposed a new anti-atherosclerosis drug (eflucimibe) for use clinically; the drug is a non-selective ACAT inhibitor. Daugherty et al. (2008. Circ. Res. 102:1445-1447) discussed macrophage accumulation at the site of atherosclerotic lesions and proposed this accumulation as a factor in growth of atherosclerotic lesions, but stated that the role for ACAT1 in macrophages had not yet been elucidated.
Hongo et al. (2009. Am. J. Physiol. Endocrinol. Metab. 297:E474-E482) focused on a link between ACAT1 gene expression in macrophages and leptin activity. The authors reported that leptin accelerates accumulation of cholesteryl ester in macrophages that is mediated by increased ACAT1 expression.
Yoshinaka et al. (2010. Atherosclerosis 213:85-91) performed in vivo studies in mice and reported that an ACAT1-specific inhibitor (K604) stimulated procollagen production independent of ACAT1 activity in macrophages, where the lesions exhibited an increase in collagen-positive areas and a decrease in macrophage-positive areas. The authors suggested that this would be a favorable plaque phenotype, indicating they provided the first evidence that an ACAT1-selective inhibitor had effects on smooth muscle cells independent of macrophage ACAT1 activity. These authors suggested that inhibiting ACAT1 might be beneficial to atherosclerosis; however, they did not demonstrate that inhibiting ACAT1, either by global inhibition or by inhibiting ACAT1 specifically in macrophages, might reduce atherosclerotic lesion, nor did they demonstrate that an ACAT1-specific inhibitor might provide benefits in obesity.
Xu et al. (2013. Acta. Biochim. Biophys. Sin. 45:953-962) disclosed that microRNAs targeted to ACAT1, and tested in vitro, reduced human ACAT1 expression and resulted in decreased macrophage foam cell formation. Another paper from the same laboratory (Huang et al. 2013. Arterioscler. Thromb. Vasc. Biol. 33:2081-2087) investigated the effect of ACAT1 inhibition as a method for treating leukemia. ACAT1 knockout mice were used. Although the focus of the paper was treatment of leukemia, and no experiments were performed in an animal model of atherosclerosis, the authors suggested that both ACAT1 and ACAT2 are targets for treating atherosclerosis. The authors proposed that partial inhibition of ACAT1 may be beneficial in atherosclerosis since total knockout of ACAT1 in mice resulted in worsening of the disease.
Various ACAT inhibitors have been shown to be effective to reduce atherosclerosis in different animal models, for either early stage or advanced stage of atherosclerosis (Rong et al. 2013. Arterioscler. Thromb. Vasc. Biol. 33:4-12; Rival et al. 2002. J. Cardiovasc. Pharmacol. 39(2):181-91; Bocan et al. 2000. Arterioscler. Thromb. Vasc. Biol. 20(1):70-9; López-Fárre et al. 2008. Cardiovasc. Ther. 26:65-74; Kusunoki et al. 2001. Circulation 103(21):2604-9), suggesting that ACAT inhibition can be a potential strategy for treating hypercholesterolemia and atherosclerosis. In these studies, however, the ACAT inhibitors administered to laboratory animals were not targeted at specific tissues. Additionally, it must be remembered that ACAT enzymes are members of the membrane-bound O-acyltransferase (MBOAT) enzyme family. MBOATs are multi-span membrane enzymes that utilize fatty acyl-CoA and a hydrophobic substance as their substrates. In humans, there are 11 MBOAT enzymes with distinct functions (Chang & Chang. 2011. Front. Biol. 6:177-182). The ACAT inhibitors used in experiments described above may produce off target effects by inhibiting other members in the MBOAT enzyme family as well.